ABSTRACT It has been more than 30 years since the first descriptions of the relationship between molecular properties of antibiotics and their ability to accumulate within gram-negative bacteria.1,2 Considerable progress towards understanding the structure and function of porin channels mediating drug uptake and RND efflux pumps involved in drug elimination has followed.3 Yet, despite these advances, concise knowledge of the rules that define small molecule accumulation within the gram-negative cell remains elusive. As such, the antibacterial drug discovery process has reached an impasse. A solution to this discovery bottleneck is desperately needed to effectively confront the threat posed by multidrug resistant gram-negative pathogens.4 Gram-negative bacteria are encapsulated by two membranes, with the asymmetric outer membrane (OM) acting as a formidable permeability barrier to small molecules, including antibacterial drugs.5 Leaving aside mechanisms of self-promoted uptake, hydrophilic antibiotics enter gram-negative cells largely through porin channels that are believed to require increased drug polarity and low molecular weight to favor passage.6?9 Yet some antibacterial drugs do not abide by these rule.10 Therefore, there must be some level of plasticity in these rules that ultimately need to be learned and exploited. Antibacterial drug discovery would greatly benefit from establishing concise rules for periplasmic accumulation through improved outer membrane penetration. Despite exhaustive screening campaigns by drug discovery companies, progress towards this goal has been limited by a number of important factors: i) few chemical classes having measurable permeability in Gram-negatives from which to derive information, ii) minimal chemical diversity among these chemical classes and iii) a general lack of broadly applicable, biologically- relevant assays to measure small molecule accumulation in MDR gram-negative pathogens. This proposal outlines a multifaceted approach to investigate determinants of small molecule permeation in gram-negative bacteria from a unique compound collection assembled at VenatoRx Pharmaceuticals as part of drug discovery programs for ?-lactamase inhibitors, Penicillin Binding Proteins. Outer membrane permeability-enabling parameters will be derived from this small molecule training set, analyzed by QSAR and Principle Component Analysis to formulate rules to guide efforts to improve periplasmic accumulation in these important pathogens. The established rule set for outer membrane penetration will be validated through direct application to an active drug discovery program for Penicillin Binding Proteins focused on Enterobacteriaceae and aiming to expand the spectrum through improved accumulation in P. aeruginosa and A. baumannii. Finally, the proposed work will focus on demystifying outer membrane permeability to relieve the bottleneck of small molecule impermeability in gram negatives and allow the rationale design of new gram-negative-biased chemical libraries to significantly improve the success rates of translating molecular screening hits into therapeutically active antibiotics.